Modified oligonucleotides, DNA probes, and their conjugates are of great value in molecular biological research and in applications such as anti-viral therapy, as probes for detecting nucleic acids, as aids in molecular biology and as pharmaceuticals or diagnostic agents. Modified oligonucleotides that can block RNA translation and are nuclease resistant are useful as inhibitors of gene expression (e.g., antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides). Oligonucleotides are important materials for research, diagnostic, therapeutic and other purposes. An ever-growing demand for improved oligonucleotides, oligonucleotide analogs and for methods for their preparation and use has arisen. Chemically modified DNA probes and their conjugates play increasingly sophisticated roles in the disparate areas of biotechnology (e.g., Barrett et al. (2003) Drug Discov Today 8, 134-141), medicine (e.g., Barrett et al., (2003) Drug Discov Today 8, 134-141), and nanotechnology (e.g., Agrawal et al., (1995) Curr Opin Biotechnol 6, 12-19). Recent chemical literature reports the synthesis of a variety of reagents and modified solid supports that allow modifications to be introduced into the structure of chemically synthesized oligonucleotides (Walton et al., (2002) Bioconjug Chem 13, 1155-1158); Niemeyer, (2002) Trends Biotechnol 20, 395-401; Ghosh et al., (2000) J Ind Chem Soc 77, 109-132). Among the many existing synthetic methods, the phosphoramidite approach is used most frequently in the synthesis of a wide variety of modified DNA probes (Sojka et al., (2000) Appl Biochem Biotechnol 89, 85-103). The discovery of the phosphoramidite method, which enables automated synthesis of natural and modified DNA molecules (Letsinger and Lunsdorf (1976) J. Am. Chem. Soc. 98:3655-3661; Caruthers et al., (1987) Methods Enzymol. 154:287-313; Beaucage and Iyer (1992) Tetrahedron, 48: 2223-2311; Protocols for Oligonucleotides and Analogs. Methods in Molecular Biology, Vol 20, Edited by Sudhir Agraval, Humana Press 1993), has stimulated the development of numerous reagents and methods to introduce a specific modification or functional group at a selected position within a synthesized oligonucleotide (Guzayev et al., (1995) Tetrahedron 51, 9375-9384; Matray et al., (1997) Bioconjugate Chem. 8:99-102; Lyttle et al., (1997) Bioconjugate Chem. 8:193-198). Some phosphoramidite labeling reagents are commercially available.
However, escalating interest in the use of chemically modified synthetic oligonucleotides in the disciplines of biology, medicine, and biotechnology (Agraval and Iyer (1999) Curr. Opin. Biotechnol. 6:12-19; Delivery Strategies for Antisense Oligonucleotide Therapeutics. Ed. Saghir Akhtar, CRC Press, 1995; Matysiak et al., (1997) Nucleosides & Nucleotides 16:855-861; Zhao et al., (2001) Nucleic Acids Res. 29:955-959) has intensified the need for less expensive and more broadly applicable labeling reagents. Despite popularity and efficiency, automated oligonucleotide synthesis cannot always address all synthetic requirements. Generally, phosphoramidite reagents used in the synthesis of modified DNA probes can be divided into two groups: A and B.
Group A phosphoramidites introduce a modification into the 5′ loci of the synthesized oligonucleotide, and group B phosphoramidites introduce a modification into the 3′ or 5′ or internal loci of the synthesized DNA molecule.
Group A phosphoramidites can be prepared with the appropriate starting compound R′ that has a hydroxyl group available for further transformations. All other functional groups of compound R′ are blocked with protecting groups that are compatible with the phosphoramidite method of oligonucleotide synthesis. The synthesis of group B phosphoramidites, however, requires preparation of an intermediate material that contains two hydroxyl groups, one of which must be selectively protected with a DMT protecting group, and one that is kept available for the phosphitylation reaction. In consequence, the preparation of group B reagents requires more labor and time-consuming synthetic effort.
Increasingly, efforts have been focused on the development of new post-synthetic strategies for the preparation of oligonucleotide conjugates with other molecules and biological moieties, as well as on new protocols for immobilizing DNA onto solid surfaces. However, flexible, effective, and efficient methods of modifying and conjugating oligonucleotides are still needed.